Browsing by Author "Wang, Hsiu-Jung"

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Polarity Alteration of a Calcium Site Induces a Hydrophobic Interaction Network and Enhances Cel9A Endoglucanase Thermostability
(2016-03)
Wang, Hsiu-Jung
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Hsiao, Yu-Yuan
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Chen, Yu-Pei
;
Ma, Tien-Yang
;
Tseng, Ching-Ping
Structural calcium sites control protein thermostability and activity by stabilizing native folds and changing local conformations. Alicyclobacillus acidocaldarius survives in thermal-acidic conditions and produces an endoglucanase Cel9A (AaCel9A) which contains a calcium-binding site (Ser465 to Val470) near the catalytic cleft. By superimposing the Ca2+ -free and Ca2+ bounded conformations of the calcium site, we found that Ca2+ induces hydrophobic interactions between the calcium site and its nearby region by driving a conformational change. The hydrophobic interactions at the high-B-factor region could be enhanced further by replacing the surrounding polar residues with hydrophobic residues to affect enzyme thermostability and activity. Therefore, the calcium-binding residue Asp468 (whose side chain directly ligates Ca2+), Asp469, and Asp471 of AaCel9A were separately replaced by alanine and valine. Mutants D468A and D468V showed increased activity compared with those of the wild type with 0 mMor 10 mM Ca2+ added, whereas the Asp469 or Asp471 substitution resulted in decreased activity. The D468A crystal structure revealed that mutation D468A triggered a conformational change similar to that induced by Ca2+ in the wild type and developed a hydrophobic interaction network between the calcium site and the neighboring hydrophobic region (Ala113 to Ala117). Mutations D468V and D468A increased 4.5 degrees C and 5.9 degrees C, respectively, in melting temperature, and enzyme half-life at 75 degrees C increased approximately 13 times. Structural comparisons between AaCel9A and other endoglucanases of the GH9 family suggested that the stability of the regions corresponding to the AaCel9A calcium site plays an important role in GH9 endoglucanase catalysis at high temperature.
Protein Thermal Stability Enhancement by Designing Salt Bridges: A Combined Computational and Experimental Study
(2014-11-13)
Lee, Chi-Wen
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Wang, Hsiu-Jung
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Hwang, Jenn-Kang
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Tseng, Ching-Ping
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生物資訊及系統生物研究所
Protein thermal stability is an important factor considered in medical and industrial applications. Many structural characteristics related to protein thermal stability have been elucidated, and increasing salt bridges is considered as one of the most efficient strategies to increase protein thermal stability. However, the accurate simulation of salt bridges remains difficult. In this study, a novel method for salt-bridge design was proposed based on the statistical analysis of 10,556 surface salt bridges on 6,493 X-ray protein structures. These salt bridges were first categorized based on pairing residues, secondary structure locations, and C alpha-C alpha distances. Pairing preferences generalized from statistical analysis were used to construct a salt-bridge pair index and utilized in a weighted electrostatic attraction model to find the effective pairings for designing salt bridges. The model was also coupled with B-factor, weighted contact number, relative solvent accessibility, and conservation prescreening to determine the residues appropriate for the thermal adaptive design of salt bridges. According to our method, eight putative salt-bridges were designed on a mesophilic beta-glucosidase and 24 variants were constructed to verify the predictions. Six putative salt-bridges leaded to the increase of the enzyme thermal stability. A significant increase in melting temperature of 8.8, 4.8, 3.7, 1.3, 1.2, and 0.7 degrees C of the putative salt-bridges N437K-D49, E96R-D28, E96K-D28, S440K-E70, T231K-D388, and Q277E-D282 was detected, respectively. Reversing the polarity of T231K-D388 to T231D-D388K resulted in a further increase in melting temperatures by 3.6 degrees C, which may be caused by the transformation of an intra-subunit electrostatic interaction into an inter-subunit one depending on the local environment. The combination of the thermostable variants (N437K, E96R, T231D and D388K) generated a melting temperature increase of 15.7 degrees C. Thus, this study demonstrated a novel method for the thermal adaptive design of salt bridges through inference of suitable positions and substitutions.